Metagenomics

As it currently stands, we do not recommend plassembler for metagenomic sequences. This is because of their high diversity, leading to difficulties in recovering chromosome-length contigs for bacteria. Additionally, Unicycler (a core dependency of Plassembler) is not recommended for metagenomes.

However, we anticipate that as sequencing becomes more accurate and cheaper, it will be increasingly possible to assemble plasmids using a plassembler like approach from metagenomes - it's a work in progress.

So as a test, we tried assembling the ZYMO HMW DNA Standard dataset from this paper, under ENA accession PRJEB48692. This mock community contains 7 bacteria and 1 fungus isolate. Notably, this dataset had extremely had deep (all bacterial chromosomes >100x coverage) and long (N50 > 20kbp) reads, so is unlikely to reflect your real-world metagenomic data as of 2023.

Get Data

# installation
mamba create -n fastq-dl fastq-dl
conda activate fastq-dl

# downloads all the read sets
fastq-dl PRJEB48692 

conda deactivate

Run Plassembler

We decided to use -m 10000, because we figures that smalll plasmids would be missed by Flye anyway, and wanted complete chromosome assemblies, and a -c 500000. We used 32 threads on 16 cores and allocated 80 GB of RAM.

plassembler run -d Plassembler_DB -l ERR7287988.fastq.gz -1 ERR7255689_1.fastq.gz -2 ERR7255689_2.fastq.gz \
-f -t 32 -q 10 -o zymo_R10.4_flye -m 10000 -c 500000

plassembler took around 8 hours (wall clock) to finish and excitingly we assembled all 7 bacterial chromosomes using Flye (unsurprising!) along with the 5 plasmids indicated in the ground truth (1 E. coli 100kbp, 1 S. enterica 49kbp and 3 small S. aureus plasmids (6, 2 and 2 kbp)) with genome fraction 100% from QUAST.

So in theory plassembler might work on metagenomes, but I would caution against using it, for now.

Contigs 34, 61, 87, 101 and 109 match what was found in the ground truth.

contig	length	mean_depth_short	circularity	PLSDB_hit	ACC_NUCCORE	Description_NUCCORE	plasmid_copy_number_short	plasmid_copy_number_long
34	110007	336.5	circular	Yes	NZ_CP061531.1	Escherichia coli strain WEM25 plasmid p1, complete sequence	1.67	1.37
61	49661	357.16	not_circular	Yes	NZ_CP012345.2	Salmonella enterica subsp. enterica serovar Choleraesuis str. ATCC 10708 plasmid pCFSAN000679_01, complete sequence	1.78	3.17
83	9628	39.11	not_circular	Yes	NZ_CP069918.1	Klebsiella oxytoca strain FDAARGOS_1334 plasmid unnamed7	0.19	0
87	6367	9554.23	circular	Yes	NZ_CP013628.1	Staphylococcus aureus strain RIVM4293 plasmid pRIVM4293, complete sequence.	47.54	12.67
91	5355	14.61	not_circular	Yes	NZ_CP068597.1	Paenibacillus sonchi strain LMG 24727 plasmid unnamed2, complete sequence	0.07	0
93	5010	13.32	not_circular	Yes	NZ_CP068597.1	Paenibacillus sonchi strain LMG 24727 plasmid unnamed2, complete sequence	0.07	0
101	2993	2561.92	circular	Yes	NZ_MH785226.1	Staphylococcus aureus strain ph1 plasmid pRIVM1295-2, complete sequence	12.75	1.65
103	2789	1018.1	not_circular	Yes	CP048737.1	Enterobacter sp. T2 plasmid unnamed1, complete sequence	5.07	4.75
106	2667	1045.66	not_circular	Yes	NZ_CP066061.1	Actinomyces oris strain FDAARGOS_1051 plasmid unnamed	5.2	4.79
108	2337	16.17	not_circular	Yes	NZ_CP069918.1	Klebsiella oxytoca strain FDAARGOS_1334 plasmid unnamed7	0.08	0
109	2216	2188.82	circular	Yes	NZ_CP013624.1	Staphylococcus aureus strain RIVM1076 plasmid pRIVM1076, complete sequence.	10.89	1.08
141	1049	1015.41	not_circular	Yes	NZ_CP066061.1	Actinomyces oris strain FDAARGOS_1051 plasmid unnamed	5.05	4.77
145	830	930.78	not_circular	Yes	NZ_CP066061.1	Actinomyces oris strain FDAARGOS_1051 plasmid unnamed